Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis

The hydrogen bonding network that surrounds the flavin in blue light using flavin adenine dinucleotide (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase from Oscillatoria acuminata (OaPAC). Our work elucidates the direct connection between BLUF photoactivation and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate the formation of the neutral flavin radical as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogues to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to <7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FAD•-. The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the adenylyl cyclase (AC) domain. While the 3F-Y6 and 2,3-F2Y6 variants undergo the complete photocycle and catalyze the conversion of ATP into cAMP, enzyme activity is abolished in the 3,5-F2Y6 and 2,3,5-F3Y6 variants where the photocycle is halted at FAD•-. Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in AC activity.

S4 Figure S1. IR difference spectra of wild-type OaPAC. Comparison of difference spectra obtained at 3 ns from TRIR (black), 100 μs from TRMPS (red) and steady state FTIR spectroscopy (blue). We observed further evolution of the protein in 1550-1650 cm -1 region. Sigma-Aldrich. The two orthogonal polyspecific aminoacyl-tRNA synthetases, E3 and E11, used to incorporate n-FY analogues were generously provided by Prof. Stubbe, MIT.

Fluorotyrosine Synthesis using Tyrosine Phenol Lyase (TPL)
The expression and purification of TPL, and the synthesis of n-FY analogues were performed using the method previously reported. 1 For this work, only the 3,5-F2Y6 and 2,3,5-F3Y6 were synthesized and purified. Briefly, TPL was expressed and purified from BL21 (DE3) pLysE Escherichia coli (E. coli), and then added to reaction mixtures containing 60 mM sodium pyruvate, 40 μM pyridoxal-5′-phosphate, 30 mM ammonium acetate, 5 mM β-mercaptoethanol and 10 mM the appropriate phenol. After 4 days, the fluorotyrosine analogues were purified, characterized using NMR and mass spectrometry, and lyophilized.

Expression and Purification of Full-Length and BLUF OaPAC
The pCold-I-OaPAC plasmid was transformed into Bl21(DE3) E. coli and grown on an LB-agar plate containing 100 µg/mL ampicillin. A single colony was used to inoculate 10 mL 2x-YT media

Incorporation of Fluorotyrosines using Orthogonal Aminoacyl-tRNA Synthetases
To extend our study to fluorotyrosine analogues with lower pKa values, 3-FY, 2,3-F2Y, 3,5-F2Y and 2,3,5-F3Y were incorporated into position 6 of full-length OaPAC. These analogues are not recognized by tyrosyl-tRNA synthetase, and instead were introduced into the protein using E3 and E11, two orthogonal polyspecific aminoacyl-tRNA synthetases. Site-directed mutagenesis was first used to generate a plasmid in which the codon for Y6 was replaced by the TAG stop codon (pCold-I-OaPAC Y6TAG) using pCold OaPAC as template, and the following primers:

Light -Dark Steady-State FTIR Difference Spectra
The steady-state spectra were obtained on a Vertex 80v (Bruker) FTIR spectrometer with difference spectrum. All measurements were performed at room temperature.

Ultrafast Time-Resolved Infrared Spectroscopy
Ultrafast time-resolved IR (TRIR) spectra were measured with ~ 100 fs time resolution at the STFC Central Laser Facility using the ULTRA apparatus described elsewhere. 2,3 TRIR spectra were acquired at 20 °C from 1400 -1800 cm -1 at a resolution of 3 cm -1 per pixel. Data were obtained using a 50 µm path length flow cell which was also rastered in the excitation beam to minimize photochemistry (photobleaching, photodegradation and photoconversion). The excitation beam of the 450 nm 100 fs 5 kHz pulses was focused to a spot size of ~ 100 μm and the pulse energy was kept below 400 nJ to avoid build-up of sample in light state. Transient difference spectra (pump on -pump off) were recorded using the IR probe at time delays between 1 ps and 2 ns. After the measurements were recorded the extent of photoconversion was shown to be negligible using absorbance spectroscopy. Spectra were calibrated relative to the IR transmission of a pure cis stilbene standard sample placed at the sample position.

Time Resolved Multiple Probe Spectroscopy (TRMPS)
TRMPS spectra were obtained at 20 °C from 100 fs to 1 ms at the STFC Central Laser Facility.
The TRMPS method has been described elsewhere, 3 and previously used by us to analyze the photoactivation of AppABLUF, and other photoactive and photochromic proteins. 1,4-6 Light sensitive samples were analyzed using a rastered flow cell, and data were acquired using a 450 nm pump pulses operated at 0.6-0.8 µJ per pulse and a repetition rate of 1 kHz. The spectral resolution was 3 cm -1 and the temporal resolution was 200 fs. A typical measurement was acquired during 45 min of data collection. All samples were prepared at 0.6-0.8 mM concentration in D2O buffer prepared with 20 mM Tris, 150 mM NaCl, pD 8.0. TRIR and TRMPS data were globally analyzed using the sequential model in Glotaran.

Ultrafast Transient Absorption experiments
Ultrafast transient absorption measurements were performed using a Spitfire Ace regenerative amplifier system providing ~800 μJ pulses centered at 800 nm at a repetition rate of 1 kHz. The output of the amplifier was split in the ratio 1:9. The pulse with the smaller energy was used for generation of the white light continuum probe in a CaF2 crystal. The higher intensity fraction was frequency doubled to 400 nm and attenuated to ~200-400 nJ/pulse before being used as the pump pulse. Polarization of the probe was again set to magic angle compared to excitation. To avoid photodegradation the samples were moved with the help of a Lissajous scanner and simultaneously flowed by a peristaltic pump. Absorption changes were measured with an Andor CCD and collected with the help of home written Labview data acquisition software, and are again reported as pump on -pump off normalized difference spectra. 7

Adenylyl Cyclase Assay
The adenylyl cyclase (AC) assay was performed using continuous and discontinuous formats which both monitored the consumption of NADH at 340 nm. In each case the assays were performed on freshly purified protein. A340 for each sample was plotted as a function of time to enable initial velocities to be extracted S18 by fitting the data to a straight line using OriginPro software. The experiment was repeated at multiple ATP concentrations (10 to 600 μM) and Vmax and Km parameters were determined by fitting the initial velocity data obtained as a function of ATP concentration to the Michaelis-Menten equation using GraphPad Prism 9 software. A similar protocol was followed for each of the OaPAC n-FY6 variants. For each protein sample, the experiment was performed in duplicates and the average of data plotted with the standard error of the mean (SEM).

Discontinuous
Continuous assay: Samples were prepared as described above except that the production of PPi was directly monitored by combining 250 µL of the pH 8.0 OaPAC/ATP reaction mixture with 125 µL of the reconstituted pyrophosphate reagent before illuminating the sample with light. The reaction mixture was incubated in a water bath at 30 °C for 1 min then 100 μL was placed in a submicro quartz cuvette. Using an Ocean Optics USB2000+ spectrometer, the absorbance at 340 nm was recorded for 40 s before the sample was illuminated using the blue LED (see above). The reaction was continuously monitored for at least 4 min and initial velocities were extracted for each ATP concentration from a plot of A340 vs time. Values for Vmax and Km were calculated by fitting the initial velocity data obtained as a function of ATP concentration to the Michaelis-Menten equation using GraphPad Prism 9. The same steps were performed for the n-FY6 variants, however for the 2,3-F2Y6, 3,5-F2Y6 and 2,3,5-F3Y6 variants a background rate in the presence of blue light and in the absence of ATP was observed, which was subtracted from the initial velocities obtained in the presence of ATP. The data reported for the continuous assay are the averaged mean of 2 replicates with their standard error of the mean (SEM). S19